Production of phenylalanine with immobilized cells

ABSTRACT

Phenylalanine is produced by contacting cells having transaminase activity with phenylpyruvic acid or phenylpyruvate in the presence of an amine donor. The cells may be ruptured or permeabilized to release their transaminase activity. Preferably, the cells are immobilized with a polyazetidine polymer. Preferred reaction conditions are an excess of amine donor in a ratio of at least 1.1:1 amine donor to phenylpyruvic acid or phenylpyruvate and a pH of 5-10 such as to convert at least 85% of the phenylpyruvic acid or phenylpyruvate to phenylalanine. Phenylalanine may also be produced from cinnamic acid using immobilized cells having phenylalamine ammonia lyase activity.

This is a continuation of application Ser. No. 518,756, filed July 29,1983, now U.S. Pat. No. 4,600,692, which is a continuation-in-part ofapplication Ser. No. 465,551, filed Feb. 10, 1983, and of applicationSer. No. 358,784, filed Mar. 16, 1982, now U.S. Pat. No. 4,436,813. Thedisclosures of these earlier applications are incorporated herein byreference.

The present invention is primarily concerned with the production ofphenylalanine from a precursor thereof, notably phenylpyruvate orphenylpyruvic acid, via transaminase. One embodiment of the inventionutilizes immobilized whole cells having transaminase activity to producephenylalanine from phenylpyruvate. However, according to a furtherembodiment of the invention, the desired enzyme activity may be obtainedby using ruptured or permeabilized cells, as such or as purifiedfractions thereof, either in the free or immobilized state to obtainphenylalanine.

The invention also contemplates the possibility of preparingphenylalanine from cinnamic acid using immobilized whole cells havingphenylalanine ammonia lyase activity.

The production of phenylalanine from phenylpyruvate has been attemptedby numerous investigators. There are two possible routes to accomplishthis transformation. One is by transamination with an appropriate aminedonor while the other is direct reductive amination using a biologicalenergy source such as NAD or NADP.

Sakurai (J. Biochemistry 43, 851, 1956) attempted the preparation ofoptically active amino acids via transamination. Sakurai used crude pigheart transaminase (freshly obtained) and found that after 20 hours theyield of phenylalanine reached a maximum of 78% when aspartic acid wasused with a small amount of glutamic acid. When aspartic acid alone wasused, the yield was only 70%. Sakurai concluded that both amino acidsshould be present for maximal yields. He explained this result as acoupled system in which glutamic acid was the amine donor forphenylalanine and the aspartic acid served to regenerate the glutamicacid.

Oishi ("The Microbial Production of Amino Acids", John Wiley & Sons, K.Yamada et al Eds. 1972, Chap. 16) reviewed the production ofphenylalanine from precursor keto acids. He noted a maximum yield of63.5% phenylalanine was obtained by Asai in screening a large number ofmicrobes which had been dried. This yield was obtained from a strain ofAlcaligenes faecalis. The two strains of E. coli surveyed showed a 38.5%and a 53% yield under the reaction conditions used. Asai obtained yieldsas high as 70.6% when the amine donor was a combination of L-aspartate,L-glutamate and L-leucine. Yields with aspartate in two-fold excess wereonly 54.5%.

It will be appreciated that the yields noted above with respect to theindicated prior procedures are not suitable for an economic industrialprocess. Yields in excess of 90% are generally considered essential fora commercially viable process.

Oishi also reported that, by using a coupled enzyme system, Kitai wasable to reach 76.8% yield. The coupled system was a yeast alcoholdehydrogenase with beef liver glutamate dehydrogenase and the Serratiamarscescens glutamate-phenylalanine amino transferase. The reaction wasdriven by the removal of acetaldehyde by semicarbazide. Additionally,Kitai was able to drive the reaction to the expected 100% yield ofL-phenylalanine by use of a coupled system for reductive amination inwhich E. coli were used to provide NADP. Glutamate, which served as theamine donor, was the limiting reagent.

Wandrey et al (U.S. Pat. No. 4,304,858) describe a coupled system (withformate dehydrogenase) for the production of phenylalanine fromphenylpyruvate while providing exogenous NAD or NADH. The system is alsoapplicable when using alphahydroxycarboxylic acid for the precursor asillustrated in U.S. Pat. No. 4,326,031. In both of these systems,however, it is necessary to use the reagent NAD or NADH and to use acoupled system in order to regenerate this expensive and labilematerial.

The available literature reviewed above indicates that only when usingcoupled systems are high yields of phenylalanine obtained fromphenylpyruvate. When uncoupled systems are used, yields no higher than71% have been obtained using three different amine donors.

An important object of the invention is to provide a process for theproduction of phenylalanine in high yields from phenylpyruvic acid orphenylpyruvate via transaminase. A more specific object is to preparephenylalanine by such a process which involves a single step, with asingle amine donor and requiring neither a coupled system nor theaddition of expensive cofactor reagents such as NADP or NAD. Otherobjects will also be hereinafter apparent.

According to the invention, phenylalanine is produced from phenylpyruvicacid or phenylpyruvate by transaminase using immobilized whole cells. Inanother embodiment of the invention, cells are used which have beenruptured or permeabilized so as to release their transaminase activity.These ruptured or permeabilized cells may be in the free or immobilizedstate.

In our above-mentioned earlier applications, we have described thepreparation and use of compositions comprising whole cells havingenzymatic activity wherein the cells are immobilized by means of aninsoluble, crosslinked polymer obtained by curing a polyazetidineprepolymer, carboxymethyl cellulose, polyurethane hydrogel prepolymer orpolymethylene isocyanate. Preferably the immobilizing polymer is apolyazetidine polymer although the other disclosed polymers may be used.Advantageously the immobilized cells are coated onto beads or otherparticulate material.

According to Ser. No. 465,551, cells may be immobilized usingpolyazetidine prepolymers which may be crosslinked in aqueous solutionby reaction with ═NH, --SH, --OH, --COOH; or other polyazetidines whichmay be crosslinked by H₂ O removal, heat, or by changing to a more basicpH. The following is an idealized structure of a representativepolyazetidine such as Polycup™ 172 (Hercules, Inc.) which is useful forpresent purposes: ##STR1## where R is typically --CH₂)₄.

The present invention contemplates the use of immobilized cellcompositions as described in said earlier applications, as well as otherforms of immobilized cells, provided the immobilized cells havetransaminase activity. According to the invention, phenylalanine isproduced by contacting a phenylalanine precursor, specificallyphenylpyruvate or phenylpyruvic acid, with the immobilized cellcomposition having transaminase activity in the presence of an aminedonor, so that the precursor is converted to phenylalanine. Theliterature indicates that small amounts of pyridoxal-5-phosphate (P5P)are required by the transaminase as a co-factor. This material (P5P) isalso used in the present process in conventional co-factor amounts.

The precursor may be used in the form of the free acid or as a saltthereof, e.g. the ammonium or alkali metal salt.

A wide variety of amine donors may be used provided these are activewith the transaminase. Preferably the donor is L-glutamic acid,L-aspartic acid or mixture thereof. However, other amine donors such asL-leucine or L-isoleucine may also give highly useful results.Preferably the donor is used in excess and it appears that higher yieldsare obtained as the excess is increased up to, for example, 30-50%excess or even more.

Any microorganism demonstrating transaminase activity may be used forpresent purposes. A wide variety of these are known (see Table 16-3,page 441, of Oishi publication mentioned above). These include thefollowing:

Alcaligenes faecalis

Pseudomonas cruciviae

Pseudomonas aeruginosa MT

Aerobacter aerogenes

Escherichia coli

Achromobacter cycloclastes

Sarcina lutea

Kluyvera citrophila

Pseudomonas fluorescens

Micrococcus lysodeikticus.

The reaction conditions used for carrying out the transaminase reactionaccording to the invention can be widely varied, as will be understoodby those in the art. For example, an aqueous solution of the precursorcan be passed through a column containing the immobilized cellscontaining transaminase activity and the amine donor. Optimum ratios ofprecursor to donor and to cells, and other operating conditions, can bereadily determined for any specific situation without undueexperimentation. Typically, however, the ratio of the amine donor to theprecursor will be at least 1:1 and preferably 1.1:1 or higher, e.g. 3:1.A preferred ratio is 1.5-2 parts donor per part precursor, parts beingon an equivalent weight basis.

Acid or alkaline pHs may be used although there will generally be areadily determined optimum pH for any particular set of conditions.Usually it is desirable to use a pH above 4, and generally one in therange of 5-10, although pHs outside these ranges may also be used.Temperatures of 30° to 40° C. normally will be used although anytemperature below transaminase deactivation can be used.

The invention is illustrated by the following examples:

EXAMPLE 1

Saccharomyces cervisiae, E. coli, Alcaligenes faecalis and Pseudomonasdachunae cells were immobilized in separate batches with polyazetidineas described in Ser. No. 465,551 (see, for instance, example 8 thereof)by mixing equal parts of cell paste and aqueous polyazetidine solution(Hercules Polycup 172), stirring to homogenity at 25° C. by hand mixingwith a wooden stick. This mixture was dispersed on Amberlite ionexchange beads which had been air-dried. The thin film ofpaste/prepolymer mixture on the beads was allowed to air dry at 25° C.One ml of each group of beads containing 0.2 grams of microbial cellsper ml of beads was then placed into a 50 ml Erlenmeyer flask containing25 ml of a 0.1M aqueous solution of sodium pyruvate and eitherL-glutamic acid, L-aspartic acid or mixture thereof as amine donor and0.1 mM of P5P. These were then compared with the use of free cells ofPseudomonas and Alcaligenes under otherwise similar conditions. Theresults in terms of phenylalanine (PHE) produced were determined by HPLCanalysis of the supernatant after 17 hours of shaking and are presentedbelow in Table I.

                  TABLE I                                                         ______________________________________                                        Transamination of Phenylpyruvic Acid                                          (PPA) To Yield Phenylalanine (PHE)                                                         Concentration PHE Formed                                                      Amine Donor:                                                     Microbe        ASP & GLU   GLU     ASP                                        ______________________________________                                        Saccharomyces cerevisiae                                                                     --          .012 M  <.002 M                                    E. coli        .024 M      .027 M   .013 M                                    A. faecalis     .01 M      .014 M  <.002 M                                    P. dacunhae    .024 M      .028 M  <.002 M                                    ______________________________________                                         (The references to "ASP" and "GLU" above represent Laspartic acid and         Lglutamic acid, respectively.)                                           

The foregoing example demonstrates that whole cells immobilized asdescribed and having transaminase activity may be effectively used toproduce phenylalanine from phenylpyruvate precursor when an appropriateamine donor is employed.

In the control using Pseudomonas and Alcaligenes in the fresh, wet freestate (unruptured and unpermeabilized), negligible transaminase activitywas noted. However, on rupturing the cells or by permeabilizing them,activity was substantially increased. This is surprising because theliterature indicates that dried cells, which would normally beconsidered lysed or permeabilized, do not give commercially acceptableconversions.

Accordingly, the use of ruptured or permeabilized cells, whetherimmobilized or in the free state, to prepare phenylalanine constitutes afurther aspect of the invention. Various techniques may be used torupture or permeabilize the cells for use according to the invention.For example, the cells may be ruptured by sonication or grinding asknown in the art. Alternatively the cells may be permeabilized bychemical treatment, e.g. by treatment with a permeabilizing surfactantsuch as Triton X100. These treatments apparently allow thephenylpyruvate or phenylpyruvic acid to more readily contact the enzymeand thus improve activity whether or not the microorganism isimmobilized.

The use of ruptured cells, and the effect of pH and amine donor level onthe results, are described in the following example:

EXAMPLE 2

2 grams of free E. coli cells were sonicated for 10 minutes in order torupture the cells after which they were incubated for 23 hours with 25ml aqueous solution containing different amounts of ASP (0.10M, 0.15Mand 0.2M), 0.1 mM P5P and 0.1M PPA at 37° C. on a Dubnoff H₂ O shaker.H₃ PO₄ was used for pH adjustment.

The results obtained are shown below in Table II.

                  TABLE II                                                        ______________________________________                                        ASP Level:                                                                            .10 M         .15 M    .2 M                                           pH      % Conversion (PPA to PHE)                                             ______________________________________                                        7       --            87       --                                             8.4     77.6          90       96.7                                           ______________________________________                                    

As shown, free cells, when ruptured, give the best and most usefulconversions at higher ASP concentrations, the data given in Table IIindicating that for a commercially acceptable yield level, the amount ofASP should exceed the amount of substrate on a molar equivalence basis.

The various aspects of the invention are further illustrated by thefollowing additional examples:

EXAMPLE 3

Table III shows the effect of pH on transaminase activity using E. coliwhole cells immobilized in bead form as in Example 1. Three experimentswere conducted using 2 ml of beads incubated in 15 ml of 0.1M PPA, 0.1mM P5P and 0.15M ASP at 37° C. for 24 hours. The pH was adjusted with 1NNaOH or 1N HCl.

                  TABLE III                                                       ______________________________________                                                   Experiment 2                                                       Experiment 1                                                                             Immobilized Cells                                                                          Experiment 3                                          Immobilized Cells  4 hours  Free Cells                                             24 hours          Activity     4 hours                                   pH   % Conv.   pH      (units)*                                                                             pH    Activity (units)                          ______________________________________                                        5.0  95.9      3        32    3      78                                       5.5  96.5      4        99    4      94                                       6.0  95.0      5       219    5     123                                       6.5  95.9      6       212    6     601                                       7.0  95.1      7       217    7     598                                       8.0  94.7      8       207    8     571                                                      9       209    8.4   586                                                      10      156    9     637                                                                     10    115                                       ______________________________________                                         *A unit is 1 micromol per hour per gram of wet cells.                    

The data in Table III shows that high yields of PHE similar to thoseobtainable with ruptured free cells can be obtained using immobilized E.coli.

EXAMPLE 4

Table IV below provides the results in terms of yield of PHE obtainedusing immobilized E. coli in a continuous column operation (300 ml,3.5×70 cm) with 0.1M PPA, 0.15M ASP and 0.1 mM P5P.

                  TABLE IV                                                        ______________________________________                                                   Experiment 1                                                                           Experiment 2                                              ______________________________________                                        DAY      1       95.1       100                                                        8       91.2                                                                  9        87.9*                                                               10       92.1                                                                 11       92.1       92                                                        12       94.2                                                                 13       95.1                                                                 14       96.1                                                                 15       94.8                                                                 16       96.8                                                                 17       95.8        85*                                                      18       95.3                                                                 35                  91                                                        42                  93                                                        43                  97                                                        45                  100                                               ______________________________________                                         *It should be noted that occasional fluctuations in flow rates may show a     reduced activity or yield on such occasions. However, the important facto     is the maximum yield which is shown as this is indicative of the full         potential of the process exemplified.                                    

EXAMPLE 5

While polyazetidine polymer is preferred for immobilizing themicroorganisms for use herein, the invention contemplates thepossibility of using any other suitable immobilizing substrate. Asrepresentative of such alternatives, there may be mentioned suchmaterials as polyacrylamide, Kappa-carrageenan, hollow fiber devices,Hypol or XAD coated beads. These materials have been shown to giveexcellent yields although the activity of the immobilized cells may varyfrom one immobilizing substrate to another. The results obtained interms of yields and activities, using different systems involvingimmobilized E. coli, are shown below in Table V. The process usedinvolved continuous flow onto a column of immobilized cells as describedof an aqueous solution of 0.1M PPA, 0.15M ASP and 0.1 mM P5P at a pH8.3-8.5 (adjusted with NH₄ OH) at 37° C. Flow varied according to columnactivity and space occupied. Equilibrium was reached at optimal flowprior to taking readings.

                  TABLE V                                                         ______________________________________                                                             Max.                                                                          Yield    Activity                                        Cell Immobilization Method                                                                         Observed (Units)*                                        ______________________________________                                        E. coli                                                                             coated on XAD beads                                                                              91       34                                                with Polycup                                                            E. coli                                                                             coated on IRA938 beads                                                                           98       63                                                with Polycup                                                            E. coli                                                                             with HYPOL foam    95       53                                          E. coli                                                                             with Kappa-carrageenan gum                                                                       100      29                                          E. coli                                                                             in a hollow fiber device                                                                         91       82                                          ______________________________________                                         *1 unit of activity is defined as 1 μ mole/hr/g cells (wet wt.) at         maximum conversion.                                                      

Of the materials referred to in Table V, XAD is a macroreticularstyrene-divinylbenzene resin; IRA 938 is an ion exchange bead resincomprising styrene-divinylbenzene containing tertiary aminesubstituents; and Hypol is a polyurethane foam. The Kappa-carrageenangum was cut into particles before use. The hollow fiber device was acommercially available item.

As an alternative to the procedures described above, phenylalanine maybe made from cinnamic acid by using immobilized whole cells which arehigh in phenylalanine ammonia-lyase activity. This aspect of theinvention represents an improvement in the process described by Yamadaet al, Applied and Environmental Microbiology November 1981, pages773-778, incorporated herein by reference.

Yamada et al describe the preparation of L-phenylalanine fromtrans-cinnamic acid by an enzymatic method using Rhodotorula glutiniscontaining L-phenylalanine ammonia-lyase activity. According to thepresent invention, Rhodotorula glutinis ATCC 10788 was grown asdescribed by Yamada et al and the harvested cells were immobilized withpolyazetidine prepolymer. 14.9 grams of cells were mixed with 14.9 gramsof polyazetidine prepolymer and coated onto 13.8 grams of IRA 938 ionexchange resin and assayed for phenylalanine ammonia lyase activity.Cinnamic acid was added to the beads by mixing 1 ml of beads produced asabove with 5 ml of assay mixture which contained 740 mg transcinnamicacid, 45 ml 28% ammonium hydroxide, pH 10 diluted to 80 ml. After 24hours the supernatant was spotted on a cellulose TLC plate and developedin a mixture of butanol, acetic acid, water (4:1:1) and the plates weresprayed with 0.2% ninhydrin and ethanol. Standards of phenylalanine wereused for comparison and an estimation based on intensity and size of thespot indicated that 0.05 mg/ml of phenylalanine had been produced.

It will be appreciated that various modifications may be made in theinvention described herein.

Accordingly, the scope of the invention is defined in the followingclaims wherein:

We claim:
 1. A process for preparing phenylalanine which comprisescontacting phenylpyruvic acid or phenylpyruvate with cells havingtransaminase activity immobilized with a polyazetidine polymer in thepresence of an excess of an amine donor in a ratio of at least 1.1:1amine donor to phenylpyruvic acid or phenylpyruvate and at a pH of 5-10,said process being such as to convert at least 85% of the phenylpyruvicacid or phenylpyruvate to phenylalanine.
 2. The process of claim 1wherein the immobilized cells are coated onto beads or other particulatematerial.
 3. The process of claim 1 wherein the excess of amine donor isat least 50%.
 4. The proces of claim 1 wherein the donor is L-asparticacid, L-glutamic acid or mixture thereof.
 5. The process of claim 1including pyridoxal-5-phosphate.
 6. The process of claim 1 wherein thecells are E. coli cells.
 7. The process of claim 1 wherein the cells areselected from the group consisting ofPseudomonas dacunhae Saccharomycescerevisiae Alcaligenes faecalis Pseudomonas cruciviae Pseudomonasaeruginosa MT Aerobacter aerogenes Escherichia coli Achromobactercycloclastes Sarcina lutea Kluyvera citrophila Pseudomonas fluorescensMicrococcus lysodeikticus.